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. Zinc binds to a site which has the same affinity for zinc as for protons. We conclude that the zinc binding site is close to a protonatable group of the bc 1 complex with pK a ‫؍‬ 7.2 which has not been identified previously. We propose that this group is part of the proton channel at the hydroquinone oxidation center of the bc 1 complex.Inhibitors have been indispensable tools for elucidating the reactions of the ubiquitous bc 1 complexes (for a review, see Ref. 1). Almost all of these inhibitors are aromatic organic compounds where at least part of the structure has some structural relationship to the substrate, ubiquinone. The single notable exception are zinc ions which have first been reported by Skulachev et al. (2) to inhibit mitochondrial respiration in micromolar concentrations. Subsequent studies (3-5) have established that the primary site of zinc inhibition in bovine heart mitochondria is the bc 1 complex.The knowledge about the bc 1 complex has largely increased over the past 20 years, including the structure of the redox centers and the peptide composition of the mammalian 11-subunit complex. The "Q-cycle" mechanism has been established for the sequence of electron and proton transfers (see Ref. 6). In the light of this body of information, we have reinvestigated inhibition by zinc to obtain information about the specific interaction between zinc ions and the bc 1 complex and to use this information to get insight into mechanistic details of the electron and proton transfer reactions. MATERIALS AND METHODSMetal salts were obtained either as chloride or as nitrate salts from Fluka. Cytochrome c (horse heart) was from Sigma, prepared without trichloroacetic acid.Bovine heart mitochondria were prepared according to Smith (7). bc 1 complex was prepared as described by Schä gger et al. (8) with the following modification: the buffer for the final Sepharose CL-6B gel filtration column was 0.05% Triton X-100, 100 mM NaNO 3 , 20 mM Pipes, 1 pH ϭ 7.2 (no NaN 3 ). Fe-S-deficient bc 1 complex ϩ antimycin was prepared following the procedure given by Schä gger et al. (9). After complete elution of the iron-sulfur protein, Fe-S-deficient bc 1 complex ϩ antimycin was eluted with 0.05% Triton X-100, 150 mM sodium phosphate, pH 7.2, and applied to a gel filtration column (Sepharose CL-6B, Pharmacia) equilibrated with 0.05% Triton X-100, 100 mM NaNO 3 , 20 mM Pipes, pH 7.2, and run with the same buffer. The fractions containing Fe-S-deficient bc 1 complex ϩ antimycin were mixed with 10% (w/v) glycerol and frozen in liquid nitrogen.Buffer for kinetic measurements was prepared as follows: 200 mM sucrose, 50 mM Pipes (from a partially neutralized stock solution), and 1 mM NaN 3 were dissolved in tridistilled water. The solution was purified over a Chelex 100 ion exchange resin column (analytical grade, Bio-Rad). 20 mM HNO 3 , 1 mM Ca(NO 3 ) 2 , and 0.024% (0.5 mM) dodecyl maltoside (Boehringer) were added, and the pH was adjusted with 2 M NaOH (purified over Chelex 100 and stored in a plastic bottle). Calcium was ...

The redox potential of the Rieske [2Fe-2S] cluster of the bcl complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25°C and I of 0.01 M was Em = f312 f 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with 14 up to Z = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k", was calculated to be approximately 2 x cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as AS" = -155 J . K-' . mol-'. The pH dependence of the midpoint potential was followed over pH 5.5 -10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH 9, indicated two deprotonations of the oxidized protein.The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pK,,,, of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Q, center of the bcl complex.The ubiquitous bc complexes are constituents of the electron-transfer chains of mitochondria, chloroplasts and bacteria. All bc complexes contain two heme-b centers, one c-type heme (heme c1 or h e m e n and a Rieske iron/sulfur protein comprising a high-potential [2Fe-2S] cluster. The bcl complexes are embedded in the respective membranes. Cytochrome b forms the core of the complex, while cytochrome cf and the iron/sulfur protein have their redox centers within the aqueous domain and are linked to the membrane part of the complex through hydrophobic anchors.The Rieske iron/sulfur protein contains, like bacterial ferredoxins, a [2Fe-2S] cluster; however, its redox potential (+ 280 mV) is approximately 700 mV more positive than that of the ferredoxins (-420 mV). Evidence has been put forward that Rieske-type [2Fe-2S] clusters, which are also present in bacterial dioxygenases, have two histidine residues ligated to the Fez+ site, in contrast to the four cysteine ligation pattern of the ferredoxins [l -41.Determination of the redox potentials of the redox centers was essential for elucidating the electron transfer pathways of the bc complexes. These studies have been performed by spectropotentiometric titration of the hemes and by EPR monitored redox titration of the iron/sulfur cluster (for a review, see [5]). A disadvantage of this approach is the use of mediators which must not affect the measured potentials [6]. EPR of the iron/sulfur cluster is only possible in frozen samples at cryogenic temperature.Recently,...